title: Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
abstract: V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μM ATP (<<Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10(6) M(-1) s(-1). At 40 mM ATP (>>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.
authors.affiliation: From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Minagawa
authors.affiliation: the Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueno
authors.affiliation: From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656.
authors.forename: Mayu
authors.initials: M
authors.lastname: Hara
authors.affiliation: the RIKEN Systems and Structural Biology Center, Yokohama 230-0045.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Ishizuka-Katsura
authors.affiliation: the RIKEN Systems and Structural Biology Center, Yokohama 230-0045.
authors.forename: Noboru
authors.initials: N
authors.lastname: Ohsawa
authors.affiliation: the RIKEN Systems and Structural Biology Center, Yokohama 230-0045.
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.affiliation: the RIKEN Systems and Structural Biology Center, Yokohama 230-0045.
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.affiliation: the RIKEN Systems and Structural Biology Center, Yokohama 230-0045.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: the Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yamato
authors.affiliation: the Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551.
authors.forename: Eiro
authors.initials: E
authors.lastname: Muneyuki
authors.affiliation: From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
authors.affiliation: the Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Chiba 263-8522, Japan. Electronic address: t.murata@faculty.chiba-u.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.affiliation: From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656,. Electronic address: iino@appchem.t.u-tokyo.ac.jp.
authors.forename: Ryota
authors.initials: R
authors.lastname: Iino
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D016983:Enterococcus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases
keywords: Bioenergetics; Enzyme Mechanisms; Molecular Motors; Single-molecule Biophysics; V0V1; V1-ATPase; Vacuolar ATPase